SUMMARY Type IV secretion systems (TFSS) are present in many different species of bacteria and play important roles both in horizontal gene transfer and in the virulence properties of several pathogens. When bacteria infect host cells, TFSS translocate proteins called ?effectors? to the target cell. The effector proteins can exert a wide variety of cell biological and biochemical changes on the host that are beneficial for the infecting pathogen. Although considerable effort has been directed at understanding the functions of many TFSS effectors, much less attention has been directed at detailed studies of the TFSS. Detailed genetic analysis of TFSS however has not been carried out in any systematic way. Genetic analysis has the potential to provide important functional information that will not come from structural studies alone. The information provided by genetic analysis will contribute to a better understanding of TFSS in several ways. The identification of permissive and non-permissive sites in TFSS components will facilitate the eventual use of biochemical tools that can be tailored to provide information about protein-protein interactions and accessibility to different types of functional probes. Identification of mutant alleles with conditional or partial phenotypes will also provide important information about the functional roles of TFSS components. We will carry out a systematic, detailed mutational analysis of permissive and non-permissive sites in the Dot/Icm TFSS components DotL, DotH< DotG, and DotE of Legionella pneumophila. This TFSS is the major virulence determinant of L. pneumophila and is capable of both effector translocation to host cells and bacterial conjugation. We will use the Mu-based Entranceposon? method to introduce 15 bp insertions (ent) into the dot/icm genes. We will screen the mutants for the ability to survive grazing by Acanthamoeba castellanii, a typical L. pneumophila host. Wild-type L. pneumophila grows within and kills A. castellanii but dot/icm Legionella mutants are digested and killed by the amoebae. Once permissive sites have been determined in the dot/icm genes, we will introduce recognition sites for TEV protease. This protease specifically recognizes a heptamer sequence that is otherwise absent in Legionella. We will study the effects of cleaving the Dot/Icm proteins that contain the TEV recognition site (TevS) in vivo under different conditions. We will also collaborate with a lab that can visualize the TFSS with Cryo-EM tomography. TEV-cleaved TFSS complexes will be examined by Cryo-EM tomography to identify the locations of specific components within the complex. This approach will allow us to determine if individual Dot/Icm components are required during translocation or if they are required for assembly of a functional TFSS but dispensable for its activity.